MEK1 Complexes regulating the timing and strength of ERK and AKT signals

Downstream of receptor tyrosine kinases and activated RAS, the RAF/MEK/ERK pathway plays major roles in cellular proliferation, differentiation, and survival. We have recently shown that one component of the pathway, MEK1, has two unexpected essential functions as a negative regulator of RAS signaling: within the pathway, it is necessary to dim ERK signaling via the MEK2/ERK axis; and outside of the ERK pathway it is required to contain the strength and duration of PIP3/AKT signaling. Crucially, both functions depend on the phosphorylation of a site present in the proline-rich region of MEK1 but not MEK2, T292. T292 is phosphorylated by activated ERK, thereby providing negative feedback to the pathway; this fact, however, also means that MEK inhibitors prevent T292 phosphorylation and therefore increase PIP3/AKT signaling. A potential consequence of this is that tumor cells will be primed for the emergence of inhibitor resistance pathways relying on AKT signaling. Negative regulation of ERK and PIP3/AKT signaling by MEK1 takes place in the context of protein complexes. We know that MEK1 negatively regulates MEK2 only if the two proteins are allowed to dimerize; and we have proven that MEK1s negative regulation of PIP3/AKT signaling requires the formation of a complex containing at least two other proteins, the large adaptor MAGI1 and the protein and lipid phosphatase PTEN. In the absence of MEK1, this complex is not formed, PTEN and MAGI1 are not recruited to the membrane, and therefore PTEN is unable to control PIP3 accumulation at the membrane. These results have shown us novel, unpredictable functions of MEK1; however, the molecular and spatial context in which these functions are carried out is still incompletely understood. Our central hypothesis is that downstream of surface receptors and Ras, MEK1 is the core of a circuitry regulating the timing and strength of ERK and AKT signals through protein/protein interaction. In this project, we will characterize the MEK1 complexes in more detail and identify interactors of MEK1 connected with its functions in restraining the ERK and AKT pathway. In particular, these will be: a) the phosphatase responsible for the removal of the RAF-dependent activating phosphorylation on MEK1 and MEK2 and thus for limiting the strength and duration of ERK signaling; and b) additional components of the MEK1/MAGI1/PTEN complex which restricts PIP3 production and therefore AKT signaling. In addition, we want to determine the subcellular localizations of these complexes to be able to correlate them with specific biological outputs.

Cell physiology can be viewed as the product of thousands of proteins acting in concert to shape the cellular response to environmental signals. These signals are interpreted by dedicated proteins that physically interact with each other to ensure to increase the intensity of the signal and maintain its fidelity. One can think of these signaling proteins as of lego bricks that can be rearranged in different shapes depending on the influence of the environment. To understand how the cell interprets the signal(s), we must be able to investigate the nature of these dynamic signaling complexes. In this project, we have produced a number of tools that will allow us to investigate the different complexes formed by the signaling protein MEK1, which is constitutively active in many human tumors, during the interpretation of signals inducing proliferation.